Insulin is a peptide hormone secreted by the β-cells of the pancreas. It consists of two peptide chains, A and B, which are linked by two intermolecular disulphide bonds. The A-chain also contains an additional intramolecular disulfide bond. Human insulin has the structure 2.

Insulin is produced as a single-chain precursor, preproinsulin, which consists of a propeptide of 24 amino acid followed by proinsulin containing 86 amino acids. The sequence of the prepropeptide is Prepeptide-[B-chain]-Arg-Arg-[connecting peptide]-Lys-Arg-[A-chain], wherein the connecting peptide consists of 31 amino acids. After the enzymatic removal of the prepeptide, the three disulfide bonds are formed and proinsulin is produced.

The mature insulin is then liberated by enzymatic cleavage of the connecting peptide at the Arg-Arg and Lys-Arg sites.
Proinsulin has a 100-fold lower affinity for the insulin receptor than native insulin because the essential residues for binding to the receptor, namely the N-terminal amino function of the A-chain and the C-terminal carboxyl function of the B-chain, are blocked.
The stability and solubility properties of insulin are important in the context of insulin therapeutics. A number of insulin analogues are known in the art.
By way of example, single-chain insulin analogues with insulin activity are disclosed in EP1193272. These single-chain insulins have a modified C-peptide of 5-18 amino acids and are reported to have up to 42% insulin activity.
U.S. Pat. No. 5,597,796 discloses insulin analogues in which two or more amino acid residues are substituted by Glu and/or Asp. Similarly, US 20090069216 and WO 2007/096332 disclose fast acting single chain insulins containing a modified B-chain and a connecting peptide. The resulting analogues are particularly well suited for transdermal administration. Fibrillation-resistant insulin and insulin analogues are disclosed in U.S. Pat. No. 8,192,957. Pegylated single chain insulins are disclosed in US 2010/0216690, whereas acylated single chain insulins are disclosed in WO 2007/104738.
WO 2005/054291 discloses single chain insulin analogues wherein the A-chain and B-chains are connected by a connecting peptide of 5-11 amino acids. Likewise, WO 95/16708 also discloses single chain insulin analogues wherein the A-chain and B-chains are connected by a connecting peptide of 1-15 amino acids, in which the C-terminal amino acid residue is other than Lys or Arg. EP0427296 discloses human insulin precursors of the general formula B(1-29)-Xn—Y-A(1-21), wherein Xn is a peptide chain with n naturally occurring amino acid residues. Similarly, EP0741188 discloses single chain insulin derivatives of the formula b-BP-a having significant insulin activity; these single chain insulins are reported to have insulin activity but also a high affinity to the IGF-1 receptor.
Insulin derivatives containing D-amino acids at their A1 position have been shown to retain their biological activity [Geiger R, Geisen K, Regitz G, Summ H D, Langner D., Hoppe Seylers Z Physiol Chem. 1980 April; 361(4):563-70]. Insulin derivatives containing D-Glu at the B21 position have been shown to be equipotent with natural insulin [Wang S H, Hu S Q, Burke G T, Katsoyannis P G. J Protein Chem. 1991 June; 10(3):313-24.] The present invention seeks to provide new single chain insulin analogues that exhibit useful therapeutic properties, wherein the two insulin chains are connected through the amino acid side chain of at least one amino acid in the insulin A-chain or the B-chain.